CN101098703A

CN101098703A - Biodegradable linkers for molecular therapies

Info

Publication number: CN101098703A
Application number: CNA2005800460377A
Authority: CN
Inventors: I·阿尔费里夫; R·J·利维; I·菲什拜因
Original assignee: Childrens Hospital of Philadelphia CHOP
Current assignee: Childrens Hospital of Philadelphia CHOP
Priority date: 2004-11-05
Filing date: 2005-11-04
Publication date: 2008-01-02
Anticipated expiration: 2025-11-04
Also published as: JP5107716B2; US8084435B2; US20070298066A1; CA2585664C; US20120141419A1; WO2006052790A2; EP1807092B1; EP1807092A4; WO2006052790A3; EP1807092A2; CN101098703B; US8580545B2; JP2008519048A; CA2585664A1

Abstract

A method and a composition for delivery of a biomaterial to an animal cell or a tissue, the composition includes (a) a biomaterial; (b) a biodegradable cross-linker portion having a hydrolyzable bond, wherein the biodegradable cross-linker portion is covalently bound to the biomaterial; and (c) a substrate, wherein the substrate is covalently bound to the biodegradable cross-linker portion, provided that the biodegradable cross-linker is adapted to hydrolyze by breaking the hydrolyzable bond and thereby release and deliver the biomaterial. A method of making the composition is also provided. A process of delivery of a biomaterial to an animal cell or a tissue is also provided.

Description

The biodegradation bridging agent that is used for molecular therapy

The cross reference of related application

The application requires the priority of the U.S. Provisional Application 60/625,386 submitted on November 5th, 2004.

Statement about the research and development of federal funding

The research part is supported by U.S. government's fund (National Heart,Lung and Blood Institute scientific research fund numbering HL72108), so U.S. government may have some right of the present invention.

Technical field

The present invention relates to finishing, more specifically relate to molecule and discharge molecule with the crosslinked of surface and when crosslinked biodegradation.

Background technology

Multiple biomaterial comprises that sending of nucleic acid, protein, cell, medicine (pharmaceuticalagent) and diagnostic agent is the focus of broad research.Gene therapy is understood to mean usually to be designed for nucleic acid is comprised that antisense DNA and RNA, ribozyme, viral genome fragment and functional activity therapeutic gene are delivered to the technology (Culver of target cell, 1994, GeneTherapy:A Handbook for Physicians, Mary Ann Liebert, Inc., New York, NY).Itself can have therapeutic activity these nucleic acid, for example suppresses the antisense DNA of mRNA translation, perhaps their human cytokines that can encode and for example promote, suppress, enlarge or replace cell function.The success of gene therapy can be weighed to the gene delivery rate that the biology that needs is arranged and the ability of amount by operation.

The carrier that the critical defect that comprises the strategies in gene therapy of stripped and vivo gene Therapeutic Method is at present described before being and the combination of delivery system can not be effectively with the cell interior of delivery of nucleic acids to target group.

It has been generally acknowledged that viral vector is the most effective delivery of nucleic acids carrier.The recombinant replication-defective viral vector has been used for external and the interior transduction of body (promptly infecting or transfection) zooblast.These carriers comprise retrovirus, adenovirus, adeno associated virus and herpesvirus vector.Though they are efficiently for gene transfer, a major defect relevant with using viral vector is that many viral vector can not infect Unseparated Cell.Another serious problems relevant with using viral genetic vector are that these carriers have the immunoreactive possibility of initiation in the patient that they are given.The effectiveness of this immunoreation limiting virus carrier is because when repeating or giving carrier for a long time, patient's immune system is removed carrier rapidly.In addition, may in cell, cause the sudden change of not expecting by inserting gene in the cytotropic genome of viral vector.The other problem relevant with viral genetic vector comprise can not be in time in transfectional cell suitably regulator gene express, have toxicity and other by sending the side effect (for example hepatic injury and myocarditis) that viral vector causes to people's tissue, and might produce and propagate harmful virion to other people.

In addition, those as using in the art methods, viral genetic vector can not be delivered to selected tissue with specific, localized mode usually.On the contrary, in tissue that a lot of prior aries method of giving viral vector is dispersed to with causing carrier system and the target tissue expected adjoins or the tissue that fluid exchange is arranged.These methods can not be located the use that viral vector has reduced these methods because non-location viral vector may transfection non-destination organization, cause immunoreation, removed in the body rapidly or have the transfection ability of decline.Be starved of existence and send the method for viral vector with locate mode.

Viral vector can be used as carrier protein and other treatment molecule is delivered in this viral vector cells transfected.These protein and other treatment molecule can be by passively, non-specificly introduce in the vector particles.Perhaps, the viral vector specificity is introduced fusion rotein, and described fusion rotein has the protein of the polypeptide virus packaging signal that is blended in wherein.

Though viral vector has been widely used in experimental gene therapy scheme and people's research (Feldman etc., 1997, Cardiovasc.Res.35:391-404; Roth etc., 1997, J.Natl.Cancer Inst.89:21-39), but still do not have any being proved to be in the viral vector mediated gene therapy effectively in these carriers.Shortcoming to the small part of supposing adenovirus vector is because the limited transgene expression that the immunoreation of host's individuality causes and the cytotoxicity of the individual organ of host caused (Smith etc., 1996, Gene Ther.3:190-200; Tripathy etc., 1996, Nat.Med.2:545-549; Nabel etc., 1995, Gene Ther.Cardiovasc.Dis.91:541-548).Other researcheres efforts be made so that the adenovirus vector sudden change is so that they have low relatively immunogenicity and toxicity.

Except the performance of most cell types to low ingestion efficiency of viral vector and the low gene construct expression of sending by viral vector, many target cell groups also exist in vivo with low quantity to make to the transfection efficiency of these particular cell types even is further reduced.Therefore, need can be used for effectively viral vector being delivered to target cell group's gene therapy method.Other work of this area for example has been devoted to attempt by the specific receptor part being connected on the carrier and with the adenovirus vector concrete cell type (Tzimagiorgis etc., 1996, Nucl.Acids 24:3476-3477) of targeting specifically.

For being used for gene delivery, viral vector must be delivered to target cell in the mode of their functions of the reservation of the biochemical composition in the viral vector.Particularly, viral vector must keep the ability that the nucleic acid that carries with the bonded ability of target cell, with carrier is transferred to cell interior, and also have the chemical reaction that catalytic nucleic acid participates in the cell in some cases ability of (for example reverse transcription, be integrated in the host cell gene group or promote the transcribing of gene element on the nucleic acid).Therefore, it is important giving the patient with viral vector and do not contact the chemically harsh or biochemical condition that goes up deactivation.In addition, many media not with contact with viral vector compatible.Ideally, viral vector is suitable for wherein or the medium on it should be biodegradable, and its form is suitable in operation intervention and the treatment intervention.

Other researcher proof by adenovirus vector is combined with polylysine or cation lipid form soluble viral carrier complexes enhancing transfection (Fasbender etc., 1997, J.Biol.Chem.272:6479-6489).But these viral complex still have many in the viral vector characteristic shortcoming described herein, and it is short to comprise that viral vector can be utilized the persistent period that contacts with desirable tissue.

A kind of method that biomaterial is sent is with containing the compositions coating medical devices of described biomaterial, and described biomaterial therefrom discharges (for example United States Patent (USP) 6,143,037 of Goldstein etc. and list of references wherein).The problem of this coating is because the character of coating materials, and they may cause acute or chronic inflammatory reaction (referring to Lincoff etc., J.Am.Coll.Cardiol., 29,808.16 (1997)).Because limited is transferred to nucleic acid among the target cell group and the ability that obtains high level expression in the gene outcome body effectively, therefore from the coating nucleic acid delivery problem is arranged also.

In addition, present method does not provide enough strong being connected between biomaterial and the delivery vector.For example, plasmid DNA introduced in the collagen sponge and, but most of DNA has just run off in the very short time (for example less than 1 hour) (referring to Bonadio etc., Nat.Med.1999,5 (7): 753-9) nucleic acid delivery successfully in its implant bone.Other known method does not provide biomaterial enough release by the mode outside the substrate biodegradation, and the biodegradation of substrate poor efficiency too.

Attempted solving these problems by in coating materials, introducing the biodegradation district.Referring to the United States Patent (USP) 6,639,014 of for example Pathak etc., it openly introduces the controlled-release delivery of the biologically active material in the biodegradation hydrogel.But this method does not solve coating materials and the problem that is connected defective tightness between the coating surface.

The present inventor had been verified in the past to be used affine conjugant (or connector) for example specific antibody or recombiant protein (as receptor fragments) is connected gene therapy vector or is contained in other delivery system (referring to the U.S. Patent application 09/487 of Levy etc. with the surface of other delivery system, 949, the United States Patent (USP) 6 of the U.S. Patent Application Publication 2003/0044408A 1 of Levy etc. and Levy etc., 333,194).

Other researcher is attempted by the following method charged biological agent being delivered to biosystem: charged biological agent is reversibly combined with the electrode surface that has opposite charges, electrode is contacted with biosystem, (for example United States Patent (USP) 4 to discharge the electric charge of electrode surface then, 585,652 and 5,208,154).Because must have electric lead that electrode is connected with power supply and the difficulty from realization from the agent of electrode surface slow-release bio, this method is seriously limited.Therefore, it is limited that these compositionss are used for viral vector is delivered to the serviceability of particular organization.

Still be starved of such compositions, it is suitable for so that the time lengthening that biomaterial is given and give relevant immunogenicity with this and reduce to minimum mode biomaterial is delivered to expectation tissue.Simultaneously, these compositionss should not influence the biologic activity (for example transfection efficiency of carrier) of the biomaterial that will send unfriendly.The compositions and methods of the invention described herein satisfy this needs.

All lists of references that this paper quotes all are incorporated herein by reference with its full content.

Summary of the invention

Therefore, the invention provides the compositions that is used for biomaterial is delivered to zooblast or tissue, described compositions comprises: (a) biomaterial; (b) has the biodegradable cross-linker part of hydrolysis key, wherein said biodegradable cross-linker part and described biomaterial covalent bond; (c) substrate, wherein said substrate and described biodegradable cross-linker part covalent bond, condition is that described biodegradable cross-linker is suitable for hydrolysis by the described hydrolysis key that ruptures, thereby discharges and send described biomaterial.

In certain embodiments, described biomaterial is the member who is selected from nucleic acid, genophore, protein, peptide and cell.In certain embodiments, described biomaterial comprises medicine.

In certain embodiments, described hydrolysis key comprises the acyl-oxygen key.

In certain embodiments, described biodegradable cross-linker partly is to be selected from following member:

In certain embodiments, described substrate is the member who is selected from metal, metal-oxide, mineral, pottery, polymer, carbon, the material that organises (organosylated material) and metallo organic material.

In certain embodiments, the described biodegradable cross-linker some effects of selection time of being enough to discharge and send biomaterial.

The present invention also provides the using method of compositions of the present invention, described method comprises: make described compositions contact a period of time with zooblast or tissue, the described time is enough to make the hydrolysis of described hydrolysis key and discharges described biomaterial, thereby described biomaterial is delivered to zooblast or tissue.In some embodiment of this method, the biodegradable cross-linker part of selecting described compositions is to influence the described time.

The present invention also provides preparation of compositions method of the present invention, and described method comprises: biodegradable cross-linker is provided, and it has the biodegradable cross-linker part that (a) contains the hydrolysis key, (b) biomaterial reaction end group and (c) substrate reaction end group; Substrate with at least one reactive group is provided; Biomaterial is provided; The substrate reaction end group reaction that makes described substrate and described biodegradable cross-linker is so that described biodegradable cross-linker part is covalently bound with described substrate; With the biomaterial reactive end radical reaction that makes described biomaterial and described biodegradable cross-linker, thereby make described biomaterial and described biodegradable cross-linker part covalently bound to prepare described compositions.In some embodiment of this method, described substrate reaction end group is the thiol reactant base.In some embodiment of this method, described biomaterial reaction end group is at least a in sulfosuccinimide base ester group, trifluoro esilate (tresylate) group and the epoxide group.In some embodiment of this method, at least one reactive group of described substrate is a mercapto.

In some embodiment of this method, described biodegradable cross-linker is to be selected from following member:

In some embodiment of this method, described biomaterial is the member who is selected from nucleic acid, genophore, protein, peptide and cell.In some embodiment of this method, described biomaterial comprises medicine.

In some embodiment of this method, described substrate is the member who is selected from metal, metal-oxide, mineral, pottery, polymer, carbon, organise material and metallo organic material.

In some embodiment of this method, described biomaterial and the reaction before described substrate and the reaction of described substrate reaction end group of described biomaterial reaction end group form the biomaterial that biodegradable cross-linker is modified.

In some embodiment of this method, biodegradable cross-linker is provided and comprises: (i) is provided first reactant with biomaterial reaction, it has the biomaterial reaction end group and first end functional groups, (ii) second reactant, its comprise biodegradable cross-linker part that (a) contain the hydrolysis key, (b) can with second end functional groups of first end functional groups reaction and (c) substrate reaction end group; Make the biomaterial reactive end radical reaction of the biomaterial and first reactant; Make first functional group and second functional group reaction, form the biomaterial that described biodegradable cross-linker is modified.

In a variant of this embodiment, described first reactant is maleimide-(sulfo group) succinimido ester, maleimide-trifluoro esilate or pyridine disulfide group-(sulfo group) succinimido ester, and described second reactant is dithiol, mercaptan-dimethyl sulfide or two (dimethyl sulfide).

The present invention also provides the method that biomaterial is delivered to zooblast or tissue, and described method comprises provides a kind of compositions, and it comprises (a) biomaterial; (b) has the biodegradable cross-linker part of hydrolysis key, wherein said biodegradable cross-linker part and described biomaterial covalent bond; (c) substrate, wherein said substrate and described biodegradable cross-linker part covalent bond, condition is that described biodegradable cross-linker is suitable for hydrolysis by the described hydrolysis key that ruptures, thereby discharges described biomaterial; With make described compositions and zooblast or tissue contact a period of time, the described time is enough to make the hydrolysis of described hydrolysis key and discharges described biomaterial, thereby and described biomaterial is delivered to described zooblast or described tissue.

The accompanying drawing summary

To describe the present invention in conjunction with the following drawings, same reference number is represented same element in the accompanying drawing, wherein:

Figure 1A is the figure that describes the biodegradable cross-linker 1 with albumino reaction sulfosuccinimide base ester group and surface reaction pyridine disulfide group.

Figure 1B is the figure that describes biodegradable cross-linker 2.

Fig. 1 C is the figure that describes the biodegradation bridging agent with trifluoro esilate (albumino reaction) base and maleimide (thiol reactant) base.

Fig. 1 D is the figure that describes the biodegradation bridging agent with epoxy (albumino reaction) base and vinyl sulfone(Remzaol (thiol reactant) base.

Fig. 1 E is the figure that describes the biodegradation bridging agent with pentafluorophenyl group ester (albumino reaction) base and iodacetyl amino (thiol reactant) base.

Fig. 2 is the synthetic figure that describes biodegradable cross-linker 1.

Fig. 3 is the synthetic figure that describes biodegradable cross-linker 2.

Fig. 4 A describes to use biodegradation bridging agent 2 with the figure of adenovirus (AdV or Ad) Covalent Immobilization on solid support.

Fig. 4 B describes to make the bridge cracking to discharge the fixedly figure of adenovirus by hydrolysis.

Fig. 5 is a rectangular histogram of describing any flat fluorescent, wherein distinguishes I and represents the GFP after 20 hours to transduce, and district II represents the GFP transduction after 72 hours, as described in example 4 above.

Fig. 6 is a curve chart of describing the aliquot release rate of accumulation of the Ad that the cross-linking agent 2 (RHC) through the cross-linking agent 1 (SHC) of hydrolysis at a slow speed and quick hydrolysis is connected.

Fig. 7 is a curve chart of describing the release of the Ad that the cross-linking agent 2 (RHC) through the cross-linking agent 1 (SHC) of hydrolysis at a slow speed and hydrolysis fast is connected, and it obtains by the surperficial fluorescence intensity of measurement in about 25 days time.

The specific embodiment

The present invention is supposed to develop such compositions and method drives, described compositions and method be used for making biomaterial (as genophore, recombinant protein, cell and medicine) and surface covalently bound so that when as described in during the compositions hydrolysis as described in biomaterial can be through the fracture of selected biodegradable cross-linker key and from the release of surface controlled.The present invention can be used for the various application that are used for biomaterial is delivered to body or cell.For example, with the new method in therapeutic virus carrier and the anti-restenosis gene therapy of the covalently bound representative of coronary artery bracket.

When research with genophore during with method that various surfaces are connected, the present inventor observes when using commercially available bi-functional cross-linking agent such as sulfosuccinimide base 6-(3 '-[2-pyridine disulfide group]-propionamido-) alkyl caproate with genophore and surperficial covalent bond, owing to carrier can not with surface isolation thereby delivery of gene successfully.This observation leads the present inventor to discovery of the present invention, wherein use the bi-functional cross-linking agent of biodegradation hydrolysis that expectation covalently bound on genophore and surface is provided, and realize that the reservation of function vector, the hydrolysis and the gene location afterwards of bridging agent shift.

Therefore, the invention provides the compositions that is used for biomaterial is delivered to zooblast or tissue, described compositions comprises: (a) biomaterial; (b) has the biodegradable cross-linker part of hydrolysis key, wherein said biodegradable cross-linker part and described biomaterial covalent bond; (c) substrate, wherein said substrate and described biodegradable cross-linker part covalent bond, condition is that described biodegradable cross-linker is suitable for hydrolysis by the described hydrolysis key that ruptures, thereby discharges and send described biomaterial.In certain embodiments, described biomaterial is nucleic acid, genophore, protein, peptide or cell.In certain embodiments, described biomaterial comprises medicine.

The present invention is based on can be covalently bound with adenovirus (Ad) and support through the cross-linking agent of cleavable, because the hydrolysis of cross-linking agent makes slow-release function Ad successfully realize the discovery of gene transfer to arterial wall.Formulate synthetic amine-mercaptan-reaction double functional cross-link agent 1 (Figure 1A) and 2 (Figure 1B) that introduce hydrolysis of ester bonds with.The t of ester linkage hydrolyzing in the model experiment proof chemical compound 1 _1/2Be several approximately weeks, the then fast order of magnitude (t of the hydrolysis of chemical compound 2 down at 37 ℃ _1/2Under similarity condition, be about several days).Therefore, the present inventor finds can control the release time of biomaterial by selecting suitable cross-linking agent.

Will be with hydrolyzable cross-linking agent 2 with mol ratio recombination adenovirus construction body Ad-GFP that modifies at 1: 30 and the rustless steel grid coupling of refining polyallylamine bisphosphonate (thiolated polyallylaminebisphosphonate) coating with monolayer thiolate.The viral vector of use fluorescent dye Cy3 labelling visually is found in physically solid (robust), the Ad layer of friction resistant (wearing and tearing) on the activatory stainless steel surfaces.In SMC culture (A10 cell), the rustless steel grid that have covalently bound Ad-GFP order about strict localized transgene expression, and it has increased X doubly in 20 to 72 hours after the transduction beginning, have reflected because the cross-linking agent hydrolysis causes the index release of Ad.Fluorescence microscope and SABC show, will form back 4 days at rat carotid artery model (n=6) with the stainless steel stent of the covalently bound similar modification of Ad-GFP, cause the transduction of a large amount of middle films and adventitia.These results confirmed by compositions of the present invention can be successfully external and send biomaterial in vivo.

To describe each component of compositions of the present invention now in detail.

Biodegradable cross-linker

Biodegradable cross-linker of the present invention comprises the biodegradable cross-linker part that (a) contains the hydrolysis key, (b) biomaterial reaction end group and (c) substrate reaction end group.Described biodegradable cross-linker can be described with following general formula:

F _t-A ¹-D-A ²-F ^p

F wherein _pBe biomaterial reaction end group, it makes the remainder (F of bridging agent _t-A ¹-D-A ²-) and aminoacid (lysine, methionine etc.) residue covalent bond; A ¹And A ²Be aliphatic series or aromatics bridge or group, it also can contain hetero atom (as O, S, NH etc.); D is degradable bridge or a group under physiological condition, and it comprises carboxylate or carbamate, or other cracked bridge of non-enzymatic gradually in water-bearing media; F _tBe the substrate reaction end group, thiol reactant base (pyridine two sulfur, maleimide, vinyl sulfone(Remzaol, iodoacetamide etc.) preferably.

In certain embodiments, described hydrolysis key comprises the acyl-oxygen key.The limiting examples of biodegradable cross-linker of the present invention is presented among Figure 1A-E, and they have with following formula (a)-(e):

Preferred cross-linking agents is formula (a) and cross-linking agent (b), and most preferred cross-linking agent is the cross-linking agent of formula (b).

In certain embodiments, described substrate reaction end group is the thiol reactant base.In certain embodiments, described biomaterial reaction end group is at least one in sulfosuccinimide base ester group, trifluoro esilate group and the epoxide group.The selection of described substrate reaction end group and described biomaterial reaction end group will be depended on the selection of the reactive group on selected surface and the biomaterial.For example, if the reactive group on described surface is a mercapto, then described substrate reaction end group is thiol reactant base such as pyridine disulfide group, maleimide amino, ethylene sulfonyl, epoxide group or iodacetyl amino.Similarly, if the reactive group of described biomaterial is amino, then described biomaterial reaction end group will be can with the group of amino reaction such as sulfosuccinimide base ester group, trifluoro esilate group, epoxide group, pentafluorophenyl group ester group etc.Substrate reaction end group and biomaterial reaction end group may be identical or may with same radical reaction, but preferred different group, and should note making substrate reaction end group and biomaterial reaction end group not react each other when selecting group, to prevent and surface and/or biomaterial reaction.Those skilled in the art do not need over-drastic experiment just should be able to select suitable group.

Two kinds of biodegradation isodigeranyl functions (amino and thiol reactant) cross-linking agent 1 and 2 (Figure 1A and 1B are respectively formula (a) and (b)) synthetic as described below with interval base of hydrolytic rupture.

Be preparation cross-linking agent 1, make the reaction of SPDP and 3-aminopropanol,, generate the acid 4 that contains ester bond with alcohol 3 acidylates of adipic anhydride with gained.Esterification by chemical compound 4 and N-hydroxysulphosuccinimide and dicyclohexylcarbodiimide (DCC) prepares cross-linking agent 1 (Fig. 2) at last.

Pyridine disulfide group-alcohol 3.

(0.76ml 10mmol) is dissolved in CH with the 3-aminopropanol ₂Cl ₂(5ml) and in the mixture of 2-propanol (3ml), in ice bath, cool off.(1.23g is 3.9mmol) at CH dropwise to add SPDP in about 1 minute ₂Cl ₂Solution (2ml).Mixture was stirred in ice bath 1.25 hours, add 13%NaH ₂PO ₄Aqueous solution (15ml) and 85%H ₃PO ₄(0.5ml).(2 * 30ml) extract organic layer 13%NaH with ethyl acetate with product ₂PO ₄, 15%KHCO ₃Washing, and vacuum drying.Crude compound 3 (1.14g) through silica gel flash chromatography purification, is used CHCl ₃Mixture (volume 100: 0-100: 7) eluting with the 2-propanol.The yield of pure compound 3: 1.01g (94%).TLC (the CHCl of chemical compound 3 ₃-2-propanol, 9: 1): a bit, R _fCa.0.3.Chemical compound 3 ¹H NMR (CDCl ₃), δ, ppm:1.68 (quintet, 6Hz, 2H), 2.60 (t, 7Hz, 2H), 3.05 (t, 7Hz, 2H), 3.4 (br., 1H), 3.42 (q, 6Hz, 2H), 3.62 (br., 2H), 6.99 (br., 1H), 7.10 (m, 1H), 7.58-7.65 (m, 2H), 8.41 (m, 1H).

Pyridine disulfide group-carboxylic acid 4.

(1.44g 5.3mmol) is dissolved in CH with alcohol 3 ₂Cl ₂(6ml), add adipic anhydride (1.74g, 13.6mmol) (preparation is referring to N.Ropson, P.H.Dubois, R.Jerome andP.H.Teyssie:Synthesis and characterization of biodegradablehomopolymers and block copolymers based on adipic anhydride.Journalof Polymer Science:Part A:Polymer Chemistry 1997,35,183-192).With the mixture vacuum drying is slurry (3.29g), makes it to react 8 hours down at 22 ℃, and dilutes with pyridine (5ml).Stir after 10 minutes, add entry (55ml), with mixture at 35-40 ℃ of following vacuum concentration to ca.30g.CHCl is used in acid 4 ₃(2 * 50ml) extractions also are extracted into 4%KHCO again ₃(in 3 * 40ml).Water layer H ₃PO ₄Be acidified to pH=3, CHCl is used in acid 4 ₃(3 * 40ml) extractions, crude compound (2.59g) is used CHCl through silica gel flash chromatography purification ₃Mixture (volume 100: 0-100: 8) eluting with the 2-propanol.Pure compound 4 yields: 1.78g (84%).The TLC:(CHCl of chemical compound 4 ₃-2-propanol, 9: 1): a bit, R _fCa.0.5.4 ¹H NMR (CDCl ₃), δ, and ppm:1.70 (m, 4H), 1.88 (quintet, 6Hz, 2H), 2.36 (t, 7Hz, 2H), 2.38 (t, 7Hz, 2H), 2.61 (t, 7Hz, 2H), 3.05 (t, 7Hz, 2H), 3.36 (q, 6Hz, 2H), 4.15 (t, 6Hz, 2H), 6.96 (br.t, 1H), 7.16 (m, 1H), 7.66-7.73 (m, 2H), 8.44 (m, 1H).

Biodegradation isodigeranyl functional cross-link agent 1

(0.934g 2.33mmol) is dissolved in the N,N-dimethylacetamide (17ml) with acid 4.Add successively N-hydroxysulphosuccinimide disodium salt (Pierce, 0.469g, 2.16mmol), (1.00g 4.85mmol) and water (2.0ml), stirs mixture 4 hours down at 20-22 ℃ dicyclohexylcarbodiimide.Filtering dicyclohexylurea precipitation, with filtrate vacuum concentration (not being higher than 0.1mmHg and not being higher than under 30 ℃) to slurry (2.5g).Divide several parts (140ml altogether) thoroughly to wash slurry with hexane, grind (45ml) to solidifying with ethyl acetate.Place under 4 ℃ and spend the night, the filtering solid is with the tert-butyl alcohol (30ml), ethyl acetate (60ml) washing, vacuum drying.Crude product 1 (1.215g) is dissolved in the methanol (30ml) and with ethanol (30ml) dilution carrying out purification, filters cellulose CC31 (Whatman) layer, to suspension (6.9g), subsequent filtration is used washing with alcohol, and vacuum drying with the filtrate vacuum concentration.Pure cross-linking agent 1 yield: 1.06g (80%).1 ¹H NMR (DMSO-d ₆), δ, ppm:1.62 (m, 4H), 1.69 (quintet, 7Hz, 2H), 2.33 (t, 7Hz, 2H), 2.49 (t, 7Hz, 2H), 2.68 (t, 7Hz, 2H), 2.85 (dd, 18,2Hz, 1H), 3.01 (t, 7Hz, 2H), 3.10 (q, 7Hz, 2H), 3.16 (br., 1H), 3.94 (br.d, 1H) 4.01 (t, 7Hz, 2H), 7.25 (m, 1H), 7.76 (m, 1H), 7.83 (m, 1H), 8.00 (br.t, 6Hz, 1H), 8.46 (m, 1H).

Be synthetic cross-linking agent 2, make beta-mercaptoethanol and 2-pyridine thionyl chloride (from 2,2 '-two pyridine disulphide and Cl ₂New system) reaction, with Boc-Gly-OSu with 2-(2-pyridine disulfide group) ethanol 5 esterifications.Boc-glycinate 6 deprotections of gained are obtained amine 7, with adipic anhydride with its acidylate.At last, to prepare cross-linking agent 2 (referring to Fig. 3) with cross-linking agent 1 similar methods.

2-(2-pyridine disulfide group) ethanol 5

With 2, (Sigma-Aldrich, 2.50g 11.35mmol) are suspended in the anhydrous pentane (150ml) 2 '-two pyridine disulphide, use Cl under 17-20 ℃ of following vigorous stirring ₂Saturated 20 minutes.The 2-pyridine thionyl chloride thickness suspension of gained is evacuated to dried under 15mm Hg, uses the argon shield residue, add anhydrous acetic acid (39ml).Continuing in 15 minutes, in stirred mixture, dropwise to add the solution of beta-mercaptoethanol (1.05ml) in anhydrous acetic acid (12ml) under 18-20 ℃ under the argon shield.Continue to stir 5 minutes, add entry (25ml).The reaction solution vacuum drying to slurry (6.66g), is added KHCO ₃(11g) solution of (65ml) in water.Product CHCl ₃(2 * 50ml) extractions, the organic layer dried over sodium sulfate leaches desiccant, and solvent removed in vacuo.Crude compound 5 (3.59g) is through silica gel flash chromatography purification, with the mixture (volume 5: 1-1: 1) eluting of hexane and ethyl acetate.The yield of pure compound 5: 2.58g (92%).5 TLC (heptane-ethyl acetate, 2: 3): a bit, R _fCa.0.4.Chemical compound 5 ¹H NMR (CDCl ₃), δ, ppm:2.93 (t, 6Hz, 2H), 3.77 (br.m, 2H), 5.75 (br.m, 1H), 7.13 (m, 1H), 7.38 (m, 1H), 7.56 (m, 1H), 8.49 (m, 1H).

The glycinate 6 of Boc-protection

Will in the alcohol 5 in the anhydrous pyridine (3.5ml) (0.818g, 4.36mmol) and Boc-glycine N-hydroxy-succinamide ester (Boc-GIy-OSu) (Sigma-Aldrich, 1.835g 6.45mmol) stirred 1 hour down at 55-65 ℃.Reactant mixture is diluted vacuum drying with toluene (30ml).Residue (3.28g) is dissolved in the ethyl acetate (40ml),, filters and wash with 10%NaCl (50ml) with hexane (100ml) dilution.The organic layer dried over sodium sulfate leaches desiccant, vacuum drying.Crude compound 6 (1.80g) is through silica gel flash chromatography purification, with the mixture (volume 5: 1-1: 1) eluting of hexane and ethyl acetate.The yield of pure compound 6: 1.41g (94%).The TLC of chemical compound 6 (heptane-ethyl acetate, 2: 3): a bit, R _fCa.0.7.Chemical compound 6 ¹H NMR (CDCl ₃), δ, ppm:1.42 (s, 9H), 3.02 (t, 7Hz, 2H), 3.88 (d, 6Hz, 2H), 4.38 (t, 7Hz, 2H), 4.98 (br., 1H), 7.08 (m, 1H), 7.60-7.66 (m, 2H), 8.45 (m, 1H).

Pyridine disulfide group-carboxylic acid 8

(1.431g 4.1mmol) is dissolved in CH with chemical compound 6 ₂Cl ₂(10ml), and add CF ₃COOH (5ml).Mixture was placed room temperature following 2 hours.Vacuum is removed volatile matter, and the trifluoro-acetate (3.59g) of the amine 7 of remnants is dissolved in CH ₂Cl ₂(10ml) and in the mixture of pyridine (5ml), in ice bath, cool off.(1.86g 14.5mmol), stirred mixture 10 minutes in ice bath, at room temperature stir 0.5h dropwise to add adipic anhydride in 1 minute.Solvent removed in vacuo with remaining slurry water (40ml) dilution, is used KHCO ₃(4.0g) neutralization, vacuum concentration is used H to 30g (removing pyridine) ₃PO ₄Be acidified to pH=3.CHCl is used in acid 8 ₃(2 * 30ml) extractions, extract dried over sodium sulfate, solvent removed in vacuo.At KHCO ₃(3.0g) exist down, in crude compound 8 (2.35g) water-soluble (60ml).Non-acidic impurities CHCl ₃-hexane (volume 3: 1,60ml) extraction, water H ₃PO ₄Be acidified to pH=4.Use CHCl ₃(after 2 * 45ml) extractions, use dried over sodium sulfate, solvent removed in vacuo is dissolved in residue (1.86g) in the ethyl acetate (4ml), and by slowly adding heptane (4ml) crystallization.Put into chemical compound 8 crystal seed assisting crystallisations.Leach crystal, with ethyl acetate-heptane (1: 1,10ml), hexane (10ml) washing, vacuum drying.The yield of pure crystalline compounds 8: 1.31g (85%).TLC (the CHCl of chemical compound 8 ₃-2-propanol, 9: 1): a bit, R _fCa.0.4.Chemical compound 8 ¹H NMR (CDCl ₃), δ, ppm:1.67 (m, 4H), 2.26 (t, 6Hz, 2H), 2.3 5 (t, 6Hz, 2H), 3.02 (t, 7Hz, 2H), 4.00 (d, 6Hz, 2H), 4.39 (t, 7Hz, 2H), 6.28 (br.t, 6Hz, 1H), 7.10 (m, 1H), 7.60-7.80 (m, 2H), 8.45 (m, 1H).

Biodegradation isodigeranyl functional cross-link agent 2.Described in above preparation cross-linking agent 1, make acid 8 (1.284g, 3.45mmol), N-hydroxysulphosuccinimide disodium salt (Pierce, 0.700g, 3.22mmol) and dicyclohexylcarbodiimide (1.50g, 4.85mmol) reaction in N,N-dimethylacetamide (26ml) and water (3.0ml).The separation of cross-linking agent 2 is similar to cross-linking agent 1 with purification.The yield of pure cross-linking agent 2: 1.652g (90%).Cross-linking agent 2 ¹H NMR (DMSO-d ₆), δ, and ppm:1.61 (m, 4H), 2.18 (t, 6Hz, 2H), 2.67 (br.t, 6Hz, 2H), 2.86 (d, 18Hz, 1H), 3.15 (br., 1H), 3.11 (t, 7Hz, 2H), 3.82 (d, 6Hz, 2H), 3.94 (br.d, 1H) 4.26 (t, 7Hz, 2H), 7.26 (m, 1H), 7.78 (m, 1H), 7.86 (m, 1H), 8.30 (br.t, 6Hz, 1H), 8.46 (m, 1H).

The functionalized surface of containing reactive group

The term that this paper is used interchangeably " surface ", " substrate ", " substrate " or " support " are meant any processing or functionalized and will handle or want functionalized to contain the surface that is adapted to pass through the functional group that biodegradable cross-linker of the present invention is connected with biomaterial.The limiting examples on described surface comprises the metal surface, have for example organic metal of the nonmetallic surface of at least one carbon and composite.

In the present invention, the even matter of solid or the heterogeneous material support of " metal support " expression homogeneous perhaps are applicable to the supporting structure network that biomaterial of the present invention is sent.The metal support can be any structure with metal surface, comprises device, and preferred medical devices.Operable any instrument, machinery or device included but not limited to surgery implant, sutures and artificial limb during the medical science that is meant term " medical devices " got involved.Be applicable to that preferably device of the present invention has the bulk of 0.1mm at least.But, also comprise littler size (promptly being lower than 0.1mm).

If device for good and all or temporarily contacts with cell or tissue, wherein an entire device or its part contact with cell or tissue, then with this device " implantation ".

The surface that the present invention is contained can have and is applicable to that various purposes for example are delivered to biomaterial organic different shape or form.Wherein, described surface can be that existing medical implant for example can functionalised the treated then support that is connected with biomaterial, cardiovascular valve or suture.In addition, can be before or after described surface modes is moulded intended shape that described surface is earlier functionalized treated then to contain described biomaterial.In some embodiments, described surface can be the form of functionalized polymer beads as described below.

Be suitable for the medical devices that biomaterial is sent among the present invention and include but not limited to cardiac valve, metal wire suture, temporary joint replacement and divulsor.Other is applicable to that medical devices of the present invention comprises orthopaedics implant such as articular prosthesis, screw, nail, nut, bolt, plate, rod (rod), pin, line, plug-in unit, osteoports, halo system and is used for stable or fixing other orthopaedics device of cutting off of vertebra and long bone fracture or joint.Other device can comprise in non-orthopaedics device, temporary substitute, permanent implant such as tracheostoma device, jejunostomy tube and gastrostomy tube, the urethra and the inlet and the contact lens of other urogenital tract implant, stylet, dilator, support, vascular clamp and filter, pacemaker, seal wire (wire guide), subcutaneous implantable intravascular intubate.In preferred embodiments, described surface is the medical devices with stainless steel surfaces, for example support.

The limiting examples that can be used for other device of the present invention comprises container, platform or the plate that can be used for for example scientific research or diagnosis.

Below further describe the example of the method that is used in functionalisation of surfaces of the present invention.

The metal surface

The metal support can be functionalized with the monomer or the polymer surfaces dressing agent that have with the chemical group of this melts combine, described in U.S. Patent application 2003/0044408 A1 of Levy of submitting on June 14th, 2002 etc., this application is incorporated herein by reference.The example of suitable metal material is rustless steel, MP35 rustless steel, aluminium oxide, platinum, platinum alloy, Elgiloy (Elgiloy), tivanium, vitallium heat-resisting alloy (vitallium), titanium, titanium alloy, Nitinol (Nitinol), chromium, cobalt and their alloy and oxide.

Be applicable to that making the functionalized coating material of metal support is any chemical compound, its (i) can carry out chemical coordination and (ii) have reactive group with the metal surface, and this reactive group is the chemical group that is suitable for carrying out with the substrate reaction end group of biodegradable cross-linker covalent reaction.

The example of described coating material includes but not limited to poly-bisphosphonate and polyamine, preferably poly-amino bisphosphonate.Other have that side chain functionalities is used for that side chain connects and the surface coordination chemical compound of amplification comprise any contain can with the group (as chelation group) of metallic ion coordination as phosphonate group, hydroxamic acid, carboxyl, sulfonyl, sulfinyl and amino polymer, oligomer or monomeric compound.

The reactive group that is used for the coating material of metal surface is the chemical group that is suitable for carrying out with the substrate reaction end group of biodegradable cross-linker covalent reaction.The limiting examples of reactive group comprises that amino or mercapto (also comprise potential modification; alkyl disulfide group for example, it can be reduced to mercapto before use), alkylation group (maleimide amino, ethylene sulfonyl, epoxide group or iodacetyl amino) and other be applicable to covalently bound other reactive group simultaneously to the group of the coordination relative inertness of lip-deep metal ion.

The polymeric skeleton of polymer surfaces dressing agent should enough be stablized in aqueous environment, can represent by the chain of forming fully the polymer of polyallylamine (for example based on) by carbon atom, perhaps can in polymeric chain (, being modified to insert the lysine residue of chelation group), introduce hetero atom (as oxygen, nitrogen etc.) for also having a part with metal-complexing better as polylysine.The polymer surfaces dressing agent can be derived from polyamine or other polymer.For example, it can be to have the side phosphonate group or together with bisphosphonate base (being used for and lip-deep metallic ion coordination) and have polymer as the alkyl disulfide group of the potential mercapto functional group that is used for subsequent reactions.

Chelation group can be by the several chemical entities that can form with the unit of metallic ion coordination, and each unitary position is close to each other, and they just can combine with same metal ion simultaneously like this, therefore increase interaction strength.Chelation group can comprise the unit (together with the bisphosphonate base, together with dicarboxylic acids ester group or company's dicarboxylic acids ester group or hydroxamic acid ester group) that can only form the metal oxygen coordination bond with metal ion, perhaps they can also comprise other atom (as the iminodiacetic acid (salt) perester radical, it can also form the metal nitrogen key that relates to uncle's amino except metal oxygen key).

Usually depend on pH with the coordination of metal surface, strong acid and strongly basic medium all suppress coordination.Stronger chelating agen (as together with the bisphosphonate base) can be used for widely pH scope (approximately from 2 to 12), and amino for wanting much weak with the coordination of metal surface, may be only effective near the very narrow pH scope of its characteristic pKa (for aliphatic amino ca.10) in.These groups are used alone or in combination the finishing that all will be suitable for based on Coordinative Chemistry.Preferably, described coating material is polyamine, poly-amino bisphosphonate, polylysine or polyallylamine.

For example, can form chemisorbed layer to combine with polyene third amino bisphosphonate (PAABP) that contains potential mercapto or poly-bisphosphonate process metal surfaces by coordination with the bisphosphonate base.If use PAABP, then the primary amino radical of PAABP chemisorbed layer can be converted into potential mercapto, it can be used for connecting biodegradable cross-linker of the present invention then.

The number of the reactive functionality that may be connected with chemisorbed layer also by several variants amplifications of using expansion chemistry (expansion chemistry).Therefore, use the amplification agent can control the number of reactive functionality.Such variant is the mercapto and the reaction that contains the polymer of a plurality of thiol reactant bases that exists on the chemisorbed layer, for example use (gather) aziridine (PEI) that 2-pyridine disulfide group (PDT yl) modifies (PEI-PDT), handle PEI-PDT formation mercapto (referring to embodiment 1) with Reducing agent then.

For example, pyridine disulfide group and mercaptan is all reactions rapidly in moisture (pH 5-8) and non-aqueous media, form stable disulfide bond.By using a large amount of excessive PAA-pyridine disulfide group polymer, most pyridine disulfide groups of amplification polymer will not react, and can be reduced the formation mercapto afterwards.Polymer with a plurality of pyridine disulfide groups can react from SPDP and polyamine such as polyallylamine and polymine and prepare.These polyamine of " free alkali " form can easily be dissolved in the nonaqueous solvent (mixture of dichloromethane or dichloromethane and isopropyl alcohol), and 0-20 ℃ down and SPDP react reposefully.Reaction is finished being less than in 30 minutes usually, does not have side reaction (degraded of the hydrolysis of succinimido ester group or pyridine disulfide group).Zhi Bei modified polymer can be by removing non-polymer impurity (N-hydroxy-succinamide and excessive sometimes SPDP) and purification with suitable solvent (as methanol or isopropyl alcohol) extraction by this way.

Nonmetallic surface with at least one carbon

The nonmetallic surface that preferably has at least one carbon is a polymer surfaces.Polymer surfaces of the present invention can be biodegradable and nonbiodegradable.The limiting examples of the polymer surfaces that uses among the present invention is polyurethane, polyester, polylactic acid, polyglycolic acid, polylactide-co-glycolide copolymer, poly epsilon caprolactone lactone, polystyrene, polyamide, rubber, silicone rubber, polyacrylonitrile, polyacrylate and polymethacrylates, poly-(alpha-hydroxy acid), poly-dioxanone, poe, polyether ester, polylactone, its mixture and copolymer thereof.

The surface (for example polymer surfaces) of containing at least one carbon can also be undertaken functionalized by the coating material that uses the reactive group that for example contains photoactivation group and expectation, wherein said photoactivation group makes dressing agent and surperficial covalent bond, makes the reactive group of expectation hang on the surface.

Term used herein " photoactivation group " expression can produce the chemical group of active substance such as free radical, nitrence, carbene and ketone activated state when absorbing external electric magnetic energy or kinetic energy (heat energy).Selected these groups produce reaction to a plurality of parts of electromagnetic spectrum, and promptly this group produces reaction to spectrographic ultraviolet, visible light and infrared part.Preferred photoactivation group is benzophenone, 1-Phenylethanone. and aromatic yl azide.When exciting, the photoactivation group can be covalently bound with surface that comprises at least one carbon such as polymer.

An example of this coating material is the water solublity light-activated polymers, the U. S. application 11/250 that continues as the present inventor's of submitting on April 16th, 2004 the PCT application PCT/US04/011861 that is entitled as " MAGNETICALLYCONTROLLABLE DRUG AND GENE DELIVERY STENTS " and the above-mentioned PCT application of submitting on October 14th, 2005, described in 877, this PCT application is incorporated herein with its full content.Described water solublity light-activated polymers also comprises the following group covalently bound with polymer precursor: the reactive group of photoactivation group, expectation and hydrophilic group based on polymer precursor.

In certain embodiments of the invention, described polymer precursor comprises the monomer that at least one is selected from allylamine, vinylamine, acrylic acid, carboxylic acid, alcohol, oxirane and hydrazides.The preferred polymers precursor is a polyallylamine.In certain embodiments of the invention, the molecular weight of polyallylamine is that about 200KDa is to about 5KDa.In preferred embodiments, molecular weight is 70KDa to 15KDa.

The reactive group of water solublity light-activated polymers is the chemical group that is suitable for the substrate reaction end group covalent reaction of biodegradable cross-linker.The limiting examples of described reactive group is mercapto, hydrazide group, epoxide group, aldehyde radical and the hydroxyl of (uncle or secondary) amino, thiol reactant base, carboxyl, mercapto, protection.Preferred thiol reactant base is the member who is selected from 2-pyridine disulfide group, 3-carboxyl-4-nitrobenzophenone disulfide group, dimaleoyl imino, iodacetyl amino and ethylene sulfonyl.

The hydrophilic group of water solublity light-activated polymers of the present invention is to be enough to that the water-soluble amount of described water solublity light-activated polymers is existed.In certain embodiments of the invention, described hydrophilic group is amino or carboxyl.

The reactive group of water solublity light-activated polymers of the present invention and hydrophilic group can be identical or different.In one embodiment of the invention, described reactive group and hydrophilic group all are amino.In another embodiment of the invention, described reactive group is a 2-pyridine disulfide group, and hydrophilic group is a carboxyl.

In certain embodiments of the invention, described photoactivation group is aryl ketones or aromatic yl azide.Preferred aryl groups ketone is benzophenone or 1-Phenylethanone..

Described water solublity light-activated polymers can have one or more photoactivation groups.In certain embodiments, described water solublity light-activated polymers per molecule has at least one photoactivation group.Preferred described water solublity light-activated polymers per molecule has a plurality of photoactivation groups.More preferably, described photoactivation group makes at least 0.1% of polymer precursor, and more preferably at least 1%, most preferably from about 20% to about 50% monomeric unit is modified.

Irradiation bomb can be the irradiation bomb that can launch the light with the wavelength that can be absorbed by photoactivation group of the present invention known in the art.With benzophenone during as the photoactivation group, preferred UV lamp.

Term used herein " water-soluble polymer " is meant that water solublity light-activated polymers of the present invention can be diluted with water at least 1 weight %, preferably single-phase to form at least 0.1 weight % under 20 ℃ temperature, and condition is water base and does not contain organic cosolvent.

In one embodiment of the invention, described water-soluble polymer is that polyallylamine is benzophenone (PAA-BzPh), and it is represented by following formula:

Wherein n is 50-2000, and k is 10-1000.

In another embodiment of the invention, described water-soluble polymer is that further to be modified the polyallylamine that contains 2-pyridine disulfide group be benzophenone (PDT-BzPh), and it is represented by following formula:

Wherein n is 50-2000, and k is 10-1000, and m is 10-1000.

When the exciting light activated group, described water-soluble polymer and surperficial covalent bond, and form monolayer from the teeth outwards.

Term used herein " layer " is meant the continuous or discontinuous deposition (deposit) that forms by polymer of the present invention and surperficial covalent bonding.Preferred described layer height is even and pure, is that it is made up of water-soluble polymer basically.

Biomaterial

Biomaterial of the present invention can be any have suitable reactive group such as carboxyl (COOH), amino (NH ₂) or mercapto (molecule SH) or macromole.For example, can use modified protein or the peptide that contains mercapto or contain amino.The thiol reactant base that is connected with a protein molecule (2-pyridine two sulfur, maleimide etc.) is widely used in preparing protein conjugate (referring to Greg T.Hermanson with the reaction of the mercapto of another protein molecule (or other biomolecule), Bioconjugate Techniques, Academic Press, SanDiego 1996).Under temperate condition, the reaction of mercapto and most of thiol reactant base (particularly 2-pyridine disulfide group) has very high specificity and very fast in water-bearing media.Can use disulphide bridges partial reduction or by use plurality of reagents make lysine residue mercaptanization with protein mercaptanization (referring to Hermanson, pp.57-70).The biomaterial that preferably has the thiol reactant base, preferred thiol reactant base be can with the degrade amino of biomaterial reactive group reaction of cross-linking agent of biotinylated biomolecule.

Described biomaterial also has treatment and uses.Suitable biomaterial comprises nucleotide sequence such as transposon; Promote the signal transducer of wound healing such as albumen such as the Bcl-1 family member and the Caspase of TGF-β, FGF, PDGF, IGF and GH albumen and adjusting cell survival and apoptosis; Tumor suppressor protein matter such as retinoblastoma, p53, PAC, DCC, NF1, NF2, RET, VHL and WT-1 gene outcome; Extracellular matrix protein such as laminin, fibronectin and integrin; Cell adhesion molecule such as cadherin, N-CAM, selection albumen and immunoglobulin; Antiinflammatory albumen such as extrasin beta-4, IL-10 and IL-12.

In certain embodiments, one of below described biomaterial comprises at least: heparin, covalency heparin or other thrombin inhibitor, hirudin, HIRULOG, argatroban, D-phenylalanyl-L-gather-L-arginyl-chloromethyl ketone or other antithrombotic, or their mixture; Urokinase, streptokinase, tissue plasminogen activator or other thrombolytic or their mixture; Fibrinolytic agent; The vasospasm inhibitor; Calcium channel blocker, nitrate, nitric oxide, nitric oxide promoter or other vasodilation; Antimicrobial or antibiotic; Aspirin, ticlopidine, glycoprotein iib/iiia inhibitor or surface glycoprotein acceptor inhibitor, or other anti-platelet agents; Colchicine or other antimitotic agent or other microtubule inhibitor, dimethyl sulfoxine (DMSO), biostearin or other secretion inhibitor agent; Cytochalasin or other actin inhibitor; Reinvent inhibitor; DNA (deoxyribonucleic acid), antisense nucleotide or other are used for the activating agent that molecular genetic is got involved; Methotrexate or other antimetabolite or antiproliferative; Citric acid tamoxifen, paclitaxel (Taxol ^TM) or derivatives thereof, or other anticancer chemotherapy medicine; Dexamethasone, dexamethasone sodium phosphate, dexamethasone acetate or other dexamethasone derivant, or other anti-inflammatory steroid or NSAID (non-steroidal anti-inflammatory drug); Cyclosporin or other immunosuppressant; Trapidil (PDGF antagonist), angiogenin, blood vessel press down the long factor antibody of peptide (growth hormone antagonist), somatomedin or antibiosis, or other growth factor antagonist; Dopamine, bromocriptine methanesulfonate, pergolide mesylate or other dopamine agonist, radiotherapeutic agents; Contain iodine compound, containing barium compound, gold, tantalum, platinum, tungsten or other heavy metal that works as radiopaque medium; Peptide, albumen, enzyme, extracellular matrix components, cell component or other biological agent; Captopril, enalapril or other angiotensin converting enzyme (ACE) inhibitor; Ascorbic acid, alpha tocopherol, superoxide dismutase, deferoxamine, the amino steroidal compounds (lasaroid) of 21-or other free radical scavenger, iron chelating agent or antioxidant; Aforementioned any material ¹⁴C, ³H, ³²P or ³⁶S radio-labeled form or other radioactive label form; Hormone; Estrogen or other gonadal hormone; AZT or other anti-polymerase agent; Acyclovir, famciclovir, rimantadine hydrochloride, Cymevan sodium or other antiviral agent; 5-amino-laevulic acid, four hydroxy phenyl chlorins, ten hexafluoro Phthalocyanine Zinc, tetramethyl hemoporphyrin, rhodamine 123 or other optical dynamic therapy agent; At Pseudomonas aeruginosa endotoxin A and with the IgG2 κ antibody of A431 epidermoid carcinoma cell effect, with the bonded monoclonal antibody of saporin, perhaps other antibody target therapeutic agent at norepinephrine energy enzyme dopamine; Gene therapeutic agents; And enalapril and other prodrug, perhaps their any mixture.Described biomaterial can also be selected from and belong to for example cell adhesion molecule of integrin, cadherin, immunoglobulin superfamily, hyaluronic acid ester receptor and mucoitin and their parts of several major receptors family.

In addition, described biomaterial can also be the right arbitrary component of any affinity ligand.The right example of this affinity ligand comprises antibiotin-biotin and IgG protein A.In addition, described biomaterial can be the right arbitrary component of any receptors ligand.An example is transferrins and receptor thereof.Other affinity ligand is to comprising powerful hydrogen bond or ionic bonding entity such as chemical complex.The latter's example comprises the metal amine complex.Other attractive complex comprises that fixedly the nucleic acid base of the oligonucleotide of particular sequence is right, and particularly antisense is right.Nucleic acid decoy (decoy) or synthetic analog also can be used as the pairing agent and combine with site interested with the genophore with design.In addition, DNA is conjugated protein also can think the specificity affinity agent; These comprise the entity such as histone, transcription factor and receptor such as glucocorticoid receptor (GR).

In a preferred embodiment, described biomaterial is anti-nucleic acid antibody.Therefore described antibody can combine with the nucleic acid specificity that coding reduces the product (or product precursor) of cell proliferation or inducing cell death, thereby alleviates the restenosis problem of tremulous pulse and other blood vessel.The nucleic acid that is connected with support through antibody is transfection/transducer cell effectively.Generally speaking, " gene therapy " field relates to sends some polynucleotide in target cell, for example the cell that contains them or organism is had antisense DNA or RNA, ribozyme, viral fragment or the functional activity gene of treatment or preventive effect.Culver，1994，GENE?THERAPY：A?HANDBOOKFOR?PHYSICIANS(Mary?Ann?Liebert，Inc.，New?York，N.Y.)。Antibody in the compositions can be the immunoglobulin molecules (as IgG antibody or IgM or any antibody subtype) of total length (being that the normal immunoglobulin gene fragment reorganization of natural existence or process forms) or immunologic competence (the being the specificity combination) part of immunoglobulin molecules.Antibody comprises one or more and the bonded site of nucleic acid specificity (being that it does not combine with the molecule of other type substantially).Binding site can be and the bonded site of the nucleic acid specificity of desired type, and irrelevant with the nucleotide sequence of nucleic acid.Perhaps binding site can be only with the bonded site of the nucleic acid specificity of the nucleotide sequence that contains expectation.Preferred antibody is the antibody that mercapto is modified.

The complex that forms between polynucleotide and the cognate antibodies can be fixed on the various surfaces, and therefore, when surface in situ contact physiological environment, the polynucleotide of connection are passed in time and are released, and its mode of sending near cell with the enhancing polynucleotide discharges.Astoundingly, connect the DNA that carries out by immunologic opsonin and shift the nucleic acid level that has kept carrying out the zone of gene therapy.

The example of suitable antibody comprises Fv, F (ab) and F (ab ') ₂Fragment, they can for example produce by handling antibody with pepsin or other protein resolvase through conventional mode.The nucleic acid binding antibody that uses in the compositions of the present invention can be polyclonal antibody or monoclonal antibody." monoclonal " antibody only comprise one type with the bonded antigen binding site of nucleic acid specificity." polyclone " antibody can comprise and the bonded a plurality of antigen binding sites of nucleic acid specificity.The antibody that uses among the present invention is full length antibody or have the antibody fragment of expectation in conjunction with character preferably, for example F (ab ') ₂

The nucleic acid that uses among the present invention can be any desired polynucleotide that are transported to cell interior.Thus, " therapeutic polynucleotide " are when offering cell or express in cell, and alleviation, inhibition or prevent disease or ill symptoms be inflammation and/or promote organization healing and the polynucleotide of reparation (as wound healing) for example.Nucleic acid can be made up of deoxynucleoside nucleic acid or ribonucleotide, can have the key of phosphodiester bond or modification, for example those of the following stated.Phrase " nucleic acid " also comprises by the typical five kinds of bases in biosystem: the polynucleotide of the base composition adenine, guanine, thymus pyrimidine, cytosine and the uracil.

Suitable nucleic acid can be linearity or cyclic DNA or RNA, and can be sub-thread or bifilar." DNA " kind of this respect comprises: eDNA; Genomic DNA; Triple helical DNA, super coiled DNA, Z-DNA and other be the DNA of common form not; The polynucleotide analog; Comprise the expression construct that coding comprises the proteic dna fragmentation of human cytokines; So-called " antisense " construct, it generates ribozyme or antisense RNA when transcribing; Viral genome fragment, for example viral DNA; Plasmid and cosmid; And gene or genetic fragment.

Nucleic acid can also be RNA, for example antisense RNA, catalysis RNA, catalysis RNA/ protein complex (i.e. " ribozyme "), and can directly be translated generation protein by it, perhaps can be inverted record and transcribe or transcribe the back translation and generate the expression construct that the RNA of RNA or protein forms respectively; Comprise and contain the transcribed construct that generates the RNA of the necessary promoter of DNA/adjusting sequence through reverse transcription; Viral RNA; And the RNA of coding human cytokines etc.Suitable nucleic acid can according to nucleic acid be delivered in the target cell or its nuclear in after will show known, expect or desired biological activity select.

The length of nucleic acid is not crucial for the present invention.Can transfection until the base pair of any number of full-length gene.For example, nucleic acid can be linearity or the ring-type distrand DNA molecule that is about 100-10000 base pair, but also can use longer or shorter nucleic acid.

Nucleic acid can be therapeutic agent, as suppressing the antisense DNA molecule of mRNA translation.Perhaps, the nucleic acid therapeutic agent of can encoding, when for example the target cell of sending when the compositions that contains nucleic acid was expressed transcription product or translation product, described transcription product or translation product had therapeutical effect for this cell or host's organism of comprising this cell.The example of therapeutic transcription product comprises protein (as antibody, enzyme, receptors bind part, wound healing albumen, anti-restenosis albumen, anticancer protein and transcribe or translate regulate albumen), antisense rna molecule, ribozyme, viral genome fragment etc.Use compositions of the present invention, nucleic acid can be encoded equally as the product of the label of cell transformed.The example of label comprises the albumen (can detect by target cell is contacted in conjunction with this proteic activating agent with specificity) of protein with discernible spectral quality such as green fluorescent protein (GFP) and cell surface expression.In addition, nucleic acid can be to be used for prophylactic preventive.

Important for the present invention nucleic acid classification comprises that coding influences the proteinic polynucleotide of wound healing.For example, gene egf, tgf, kgf, hb-egf, pdgf, igf, fgf-1, fgf-2, vegf, other somatomedin and receptor thereof are brought into play important effect in wound healing.

The polynucleotide of the factor of another kind of coding and regulating or opposing inflammatory process also are important for the present invention.In addition, the gene of the receptor antagonist of coding antiinflammatory such as MSH, cytokine such as the IL-10 or the reaction that reduces inflammation also is a dependency.

Can encode inducing cell death or promote the expression product of cell survival of suitable polynucleotide, this depends on this nucleic acid.These polynucleotide are not only useful for treatment carcinogenic cells and other abnormal cell, and also useful for inducing the apoptosis in the normal cell.Therefore, another noticeable nucleic acid classification of the present invention relates to a kind of polynucleotide, coding anticancer protein or produce anticancer antisense oligonucleotide when transcribing when it is expressed.Thus; phrase " anticancer protein " and " anticancer antisense oligonucleotide " represent respectively when before the carcinous infringement in zone that provides it to any desired cell death or the individuality or the cancer during damaging part, its in this position prevention, suppress, reverse the albumen or the antisense oligonucleotide of unusual or normal cell growth or cell death inducing.According to the present invention, these polynucleotide are delivered to cell can cell growth inhibiting, differentiation or migration to be to prevent metastasis site or near moving or not desirable tissue expansion it.The example of this anticancer classification is the polynucleotide of one of known antitumor protein of coding.These polynucleotide for example will comprise from or derived from one or more the nucleotide sequence in the following gene: ab1, akt2, apc, bcl2-a, bcl2-β, bcl3, bcl3, bcl-x, bad, bcr, brca1, brca2, cb1, ccnd1, cdk4, crk-II, csflr/fms, db1, dcc, dpc4/smad4, e-cad, e2fl/rbap, egfr/erbb-1, elk1, elk3, eph, erg, ets1, ets2, fer, fgr/src2, fas, fps/fes, fra1, fra2, fyn, hck, hek, her2/erbb-2/neu, her3/erbb-3, her4/erbb-4, hras1, hst2, hstf1, ink4a, ink4b, int2/fgf3, jun, junb, jund, kip2, kit, kras2a, kras2b, ck, lyn, mas, max, mcc, met, mlh1, mos, msh2, msh3, msh6, myb, myba, mybb, myc, myc11, mycn, nf1, nf2, nras, p53, pdgfb, pim1, pms1, pms2, ptc, pten, raft, rbl, re1, ret, ros1, ski, src1, tal1, tgfbr2, thra1, thrb, tiam1, trk, vav, vh1, waf1, wnt1, wnt2, wt1 and yes1.In like manner, suppress the oligonucleotide that one of these genes express and can be used as anticancer antisense oligonucleotide.

Nucleic acid with key between the nucleotide of modification also can be used in the compositions of the present invention.For example, can use the nucleic acid of key between the nucleotide that contains modification, the nuclease stability that its performance increases.These polynucleotide comprise and for example contain one or more phosphate esters, thiophosphate, phosphorodithioate, phosphoramidate, methoxy ethyl phosphoramidate, dimethoxym ethane (formacetal), sulfur dimethoxym ethane (thioformacetal), diisopropyl silicyl, acetamidate, carbamate, dimethylene sulfide (CH ₂-S-CH ₂-), dimethylene sulfoxide (CH ₂-SO-CH ₂-), dimethylene sulfone (CH ₂-SO ₂-CH ₂-), 2 '-O-alkyl and 2 '-deoxidation-2 '-fluoro-thiophosphate nucleotide between the polynucleotide of key.

Be purpose of the present invention, nucleic acid can be generally used for preparing or the conventional method of isolating nucleic acid is prepared or separates by any.For example, use reagent and the synthesizer chemical synthesising DNA and the RNA of commercially available acquisition according to known method.For example referring to Gait, 1985: OLIGONUCLEOTIDE SYNTHESIS:A PRACTICAL APPROACH (IRL Press, Oxford, England) in.The RNA molecule also can use plasmid as can (Madison, WI) SP65 of Huo Deing prepares with high yield from Promega Corporation by the in vitro transcription technology.Nucleic acid can be by any suitable method purification, and many such methods all are known.For example, nucleic acid can pass through reversed-phase HPLC or ion exchange HPLC, molecular-exclusion chromatography or gel electrophoresis purification.Certainly, those skilled in the art will recognize that purification process will partly depend on the size of the DNA that wants purification.Nucleic acid can also be by any preparation the in the countless versions recombinant technique of known or later exploitation.

Suitable nucleic acid can be introduced in the various known host carrier systems through genetic engineering, and this system is provided for to be suitable for preparing the scale replicating nucleic acid of invention compositions.Carrier system can be virus or non-virus carrier.The instantiation of virus carrier system comprises adenovirus, retrovirus, adeno-associated virus and herpes simplex virus.The preferred adenovirus vector that uses.The non-virus carrier system comprises plasmid, ring-type distrand DNA molecule.Virus and non-virus carrier system can use the known method design to be used to instruct the necessary element of the nucleic acid of transcribing, translating or transcribe and translate to comprise in the cell of sending at it.Can use method known to those skilled in the art to make up to have operationally the expression construct of the protein coding sequence that is connected with suitable transcribing/translate control signal.These methods comprise extracorporeal recombinant DNA technology and synthetic technology.For example, referring to Sambrook etc., 1989, MOLECULAR CLONING:A LABORATORYMANUAL (Cold Spring Harbor Laboratory, New York) and Ausubel etc., 1997, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (JohnWiley ﹠amp; Sons, New York).

The nucleic acid of one or more proteins of interest of encoding operably links to each other with various promoter/regulon sequence.Promoter/regulon sequence can comprise composing type or inducible promoter, can use high level expression or regulating and expressing to instruct gene of interest under suitable condition.The instantiation in operable promoter/regulon zone comprises cytomegalovirus (CMV) promoter/adjusting subarea and the promoter/adjusting subarea relevant with SV40 early gene or SV40 late gene.Preferred end user CMV promoter, but any promoter/adjusting subarea of instructing gene of interest high level expression or regulating and expressing can be used substantially.

Employed nucleic acid contains a plurality of protein-coding regions of being combined on the individual gene construct under one or more promoteres controls also within the scope of the invention.Two or more protein-coding regions can be the transcribing under the control of single promoter, and transcribing of nucleic acid can comprise one or more internal ribosome entry sites between protein-coding region.Therefore, can use different gene of countless versions and genetic constructs.

Biomaterial of the present invention also comprises medicine, photographic developer and diagnostic agent.

In some embodiment of described compositions, described biomaterial is selected from antibody, viral vector, somatomedin, biologically active polypeptide, the polynucleotide of encoding human active polypeptide, cell is regulated micromolecule, peptide, albumen, oligonucleotide, gene therapeutic agents, the gene transfection carrier, receptor, cell, medicine, the medicine delivery agents, nitric oxide, antimicrobial, antibiotic, antimitotic agent, dimethyl sulfoxine, the secretion inhibitor agent, the anticancer chemotherapy medicine, steroidal and NSAID (non-steroidal anti-inflammatory drug), hormone, extracellular matrix, free radical scavenger, iron chelating agent, antioxidant, photographic developer, radiotherapeutic agents.Preferably, described biomaterial is anti-knob antibody, adenovirus, COxsackie adenovirus receptor D1 territory (CAR D1), insulin, angiogenesis peptide, anti-angiogenic peptides, avidin, biotin, IgG, a-protein, transferrins and TfR, cell adhesion molecule and cell adhesion molecule part.In some embodiment of this method, described biomaterial is selected from the member of nucleic acid, genophore, albumen, peptide and cell.Several different biomaterials can be fixed on the same surface.

Preparation of compositions method of the present invention

The present invention also provides described preparation of compositions method.This method comprises provides biodegradable cross-linker, substrate with at least one reactive group is provided, biomaterial is provided, the substrate reaction end group reaction that makes described substrate and described biodegradable cross-linker is so that the biodegradable cross-linker part is covalently bound with substrate, and the biomaterial reactive end radical reaction that makes described biomaterial and described biodegradable cross-linker, thereby make described biomaterial and described biodegradable cross-linker part covalently bound to prepare described compositions.The limiting examples of this method is presented among Fig. 4 A.Be not at first to make described biomaterial and the reaction of described cross-linking agent, but as if this is more easily in proper order.Therefore, in certain embodiments, the described biomaterial reactive end radical reaction that makes described biomaterial and described biodegradable cross-linker is before the described substrate reaction end group reaction that makes described substrate and described biodegradable cross-linker or carry out simultaneously with it.

In some embodiment of this method, described biodegradable cross-linker is provided and comprises: (i) is provided first reactant with the reaction of described biomaterial, it has the biomaterial reaction end group and first end functional groups, (ii) second reactant, it comprises the biodegradable cross-linker part that (a) contains the hydrolysis key, (b) can with second end functional groups of first end functional groups reaction and (c) substrate reaction end group; Make the biomaterial reactive end radical reaction of the biomaterial and first reactant; With make first functional group and second functional group reaction, form the biomaterial that described biodegradable cross-linker is modified.In these embodiments, described biodegradable cross-linker directly is formed on the biomaterial, and the biomaterial/cross-linking agent combination with gained is connected with the surface then.In a variant of this embodiment, described first reactant is maleimide-(sulfo group) succinimido ester, maleimide-trifluoro esilate or pyridine disulfide group-(sulfo group) succinimido ester, and described second reactant is dithiol, mercaptan-dimethyl sulfide or two (dimethyl sulfide).

Can select according to desired results such as conditions such as temperature, buffer agent and reaction materials.Can carry out this method according to general chemical principle those skilled in the art.Below the limiting examples of this method further is provided in embodiment 2-4.

Biomaterial is sent

The present invention also provides the using method of compositions of the present invention, the delivering method of biological example material, described method comprises: make described compositions contact a period of time with zooblast or tissue, this time is enough to make the hydrolysis of described hydrolysis key and discharges described biomaterial, thereby described biomaterial is delivered to zooblast or tissue.The limiting examples of this method shows in Fig. 4 B.Embodiment 5 has described an example of this method.

In some embodiment of this method, the biodegradable cross-linker of selected compositions influences the described time.As previously mentioned, biodegradable cross-linker is hydrolysis or slow hydrolysis comparatively fast, and this depends on its design.Therefore, delivery rate can be controlled by selecting suitable biodegradable cross-linker.In addition, depend on the number of the reactive group on upward surperficial and the biomaterial, the amount that is connected lip-deep biomaterial can change.For example using, the amplification method of PEI-PDT can be used for obtaining to contain expected response radix purpose surface.In addition, when handling PDT group and mercapto exchange when further react with biodegradable cross-linker, can be with a part of PDT group rather than whole PDT base group modifications, so the method for the biomaterial load capacity that another kind of selection expects is provided again.According to this guidance, the load capacity of biomaterial can be chosen as can utilize 100% to 0.1% of group.In a variant, load capacity is 25%.

With reference to further illustration the present invention in detail of following examples, still should be appreciated that and do not think that the present invention is limited to this.

Embodiment

Embodiment 1

Originally experimental results show that the isodigeranyl functional cross-link agent sulfosuccinimide base 6-that uses the mercaptan cleavable (3 '-[2-pyridine disulfide group]-propionamido-) the alkyl caproate ((PierceBiotechnology of sulfo group-LC-SPDP), Inc., Rockford is IL) with biomaterial and the direct covalently bound idea of stainless steel surfaces.

Make the adenovirus (Cy3Ad-GFP) (batches 11 of 500 μ lCy3-labellings; 1.3 * 10 ¹²Granule) at room temperature reacted 70 minutes with 15mg LC-sulfo group-SPDP, generate the adenovirus (Cy3Ad-PDT-GFP) of the DPT modification of Cy3-labelling.It is the SLIDE-A-LYZER dialysis cassette of 10kDa that reactant mixture is placed cutoff value, dialyses 22 hours with PBS, changes PBS three times.With the last a PBS degassing, it contains 10mM EDTA, and dialysis is carried out under argon atmospher.

Second day, by standard method (promptly contacted 1N nitric acid 15 minutes, contacted isopropyl alcohol then 15 minutes) 9 316L steel sieves of pretreatment with distilled water washing 5 times.

With 6 sieves incubation 5 hours in the PrSSPAABP solution 1.3% under 60 ℃; described PrSSPAABP is with 2; the polyallylamine (PAA) that 2-two phosphono ethyls (BP) and pyridine disulfide group (PrSS) are modified; and 3 sieves under 60 ℃ with 2, incubation is 5 hours in 3% polyallylamine (PAABP) solution that 2-two phosphono ethyls are modified.To sieve then with distilled water (DDW) washing, and 6 sieves that use that PrSSPAABP handles and three (2-carboxy ethyl) phosphines (TCEP) (20mg/ml is at the 0.1M acetate buffer) at room temperature reacted 25 minutes.Then, with acetate buffer and PBS washing sieve, (PEI-PDT) increase agent (high degree of modification (aziridine key sum 25%) 09 IA-46-4-0.5ml, DDW-1.25ml, 0.4M acetate buffer-0.25ml) reacted 40 minutes of 3 sieves and the polymine (PEI) of modifying wherein at RT with under jolting with 2-pyridine disulfide group (PDT yl).The purpose of amplification program is to increase the number that the PDT group is gone up on the surface.

React with other 3 sieves and with the adenovirus (Cy3Ad-PDT-GFP) that the DPT of the Cy3-labelling of 3 sieves of PAABP incubation, the 150 μ l dialysis degassing in little Eppendorf pipe and in 4%BSA modifies.3 sieves that react with the amplification agent wash with acetate buffer and PBS, and react 20 minutes with jolting down in room temperature with DTT (20mg/ml is in DDW).At last, will sieve washing, the Cy3 Ad-PDT-GFP reaction of the 150 μ l dialysis degassing in little Eppendorf pipe and in 4%BSA with PBS.For all 3 groups, virus is combined with sieve spend the night.

Generally speaking, the chemical scheme of 3 groups of sieves can followingly be represented:

1. matched group: Me-PAABP+ (PDT-Ad) (no covalent bond form)

2. there is not the amplification group: (Me-PrSSPAABP+TCEP)-＞Me-PAABP-SH+PDT-Ad-＞Me-PAABP-Ad

3.PEI amplification group: (Me-PrSSPAABP+TCEP)-＞Me-PAABP-SH+PDT-PEI-＞Me-PAABP-PEI-PDT (n)+DTT-＞Me-PAABP-PEI-SH (n)+PDT-Ad-＞Me-PABPP-PEI-Ad

In this general approach,, Cy3Ad-PDT-GFP is abbreviated as PDT-Ad for making things convenient for reference.

Second day, test sieve under fluorescence microscope.Substantially do not observe fluorescence in the control sample (the 1st group), and in the 2nd group sample, observe minuent, in the 3rd group of sample, observe moderate to height fluorescence to moderate fluorescence.

On the same day, HEK 293 and A 10 (rat artery smooth muscle cell) cell culture that sieve is placed ca.60% converge.Sieve is placed back 24 hours each Kong Zhongjun and is not observed any GFP positive cell.Changing culture medium then, is that the DTT of 40mg/ml is dissolved in the culture medium (the every hole of 80mg/) with concentration.The GFP positive cell did not appear in 5 days in incubation yet in addition.This covalently bound dose of experimental results show that difficult degradation is as sulfo group-LC-SPDP prevention adenovirus transduction.

Embodiment 2

Carry out this experiment and detect Ad and the surperficial covalently bound strategy of steel with (hydrolyzable) N-succinimido 3-(2-pyridine disulfide group)-propionic ester (SPDP) cross-linking agent of the cleavable that has the long time hydrolysis dynamics in use.With 12 316L steel sieves of standard method pretreatment, wherein 8 sieves reacted 5 hours under 60 ℃, in 1%PrSSPAABP.4 contrast sieves (do not have mercapto on the PAABP, thus not with the Ad covalent bonding) 60 ℃ down with 2,3% polyallylamine (PAABP) that 2-two phosphono ethyls are modified reacted 5 hours.Wash sieve, the sample and the TCEP (20mg/ml is in the 0.1M acetate buffer) that handle with PrSSPAABP reacted 25 minutes in room temperature with under jolting.After the TCEP cracking, will sieve in DDW and wash, and with PEI-PDT amplification agent (09IA-46-4; 0.5ml, DDW 1.25ml, 0.4M acetate buffer 0.25ml) reaction 1 hour.Wash sieve with acetate buffer and DDW then, make it to react 20 minutes with jolting down in room temperature with DTT (20mg/ml is in DDW).

Abreast, making the SPDP analog of 750 μ l Cy3AdGFP (batch 14) and 30mg cleavable is that biodegradable cross-linker 1 (12IA-40-1) (seeing figure IA) is in room temperature with jolt reaction down 1 hour.The virus that use is modified through the degassing pretreated desalting column of PBS/EDTA (Ultragel A6) purification.

Activatory virus and mercaptan sieve and contrast sieve are reacted in 5%BSA/ degassing PBS to spend the night.Second day, test sieve under fluorescence microscope.Contrast and suitable bonded sieve all show Cy3Ad and exist.But, the amount of relevant Ad much higher (term " suitably bonded " is meant that wherein Ad is by the mercapto and the in fact covalently bound situation of sieve of reduction PrSSPAABP) in the suitable bonded sieve.Carry out this contrast to estimate and the non-specific binding component of sieving in bonded total precursor virus.

A10 cell culture with sieve places the HEK 293 and 30% that converges to converge detects the transduction situation after 24 hours.Place the control sample of HEK 293 and A10 cell and the cell that suitable bonded sample is not all observed transduction.Therefore, use the connection dosage that uses in the present embodiment, the long time hydrolysis dynamics of bridging agent can not make adenovirus transduce.

Embodiment 3

Carry out this experiment and make the covalently bound strategy of Ad and steel surface to probe into (hydrolyzable) SPDP cross-linking agent that uses cleavable with quick hydrolysis dynamics.

12 316L stainless steel sifts are cleaned with isopropyl alcohols and 1N nitric acid, at 80 ℃ with acutely jolted time in 1.5%PrSSPAABP incubation 3.5 hours.With DDW washing sieve, make it and TCEP (30mg/ml; In the 0.1M acetate buffer) reacted 30 minutes in room temperature with under jolting.After using acetate buffer and water washing successively, make sieve and PEI-PDT at 42 ℃ with acutely jolt down and reacted 90 minutes.Reacted 30 minutes in room temperature with under acutely jolting then with PBS washing sieve, and with DTT (25mg/ml is in DDW).

Abreast, with 0.5ml carbonate buffer (pH=9.3) dilution 1mlAd-GFP (self-produced batch of material, 2.25 * 10 ¹²/ ml).The cross-linking agent 2 (13IA-55-3) (seeing Figure 1B) of the rapid hydrolysis of 12.2mg is dissolved among the 1ml PBS, and this solution of 64 μ l is added in the Ad suspension.Again 1mg Cy3-NHS dyestuff is dissolved in the 1ml carbonate buffer, this diluent of 200 μ l is added in the reactant mixture.Make to be reflected at room temperature and moderate and to jolt down and carry out 40 minutes, under 4 ℃, carried out 30 minutes again.Use then Ultragel A6 with the Ad gel filtration to the PBS/EDTA of the degassing, merge the part (5-7.5ml) contain virus, and blast argon.The two Ad that modify of 1ml with 1ml degassing PBS dilution, are made it and sieve argon atmospher, 22 ℃ and acutely jolt (240rpm) reaction 14 hours down.

Second day, with the fluorescence microscopy sieve, observe the strong viral bonded surface marker that shows, and free two Ad that modifies only has very faint fluorescence.Astoundingly, lip-deep Ad layer is very wear-resistant, and fluorescence is still most of when sieving keeps because the rubber gloves that is soaked in water is rubbed.

2 sieves are placed HEK 293 cell cultures, 4 sieves are placed A 10 cell cultures.All the other 4 screenings do not place the Eppendorf pipe of 250 μ l PBS, with test tube at room temperature (n=2) or under 4 ℃ (n=2) jolt 68 hours.

Sieve was placed back 18 hours, all observed strong location transduction in HEK 293 and A 10 cultures.To be transferred to from the sieve of A10 culture then in the new cell culture, and observe strong new transduction (not shown) after 18 hours, this shows that the carrier that continues survival still exists.

The designated sieve that is used for external " release " experiment was analyzed behind the beginning incubation in 68 hours.Compare with the sieve of at room temperature handling at the sieve of 4 ℃ of following incubations and to have kept the more Ad of the Cy3 labelling of a large amount.This observe with hypothesis based on Ad is consistent with the releasing mechanism of the hydrolysis of the surperficial cross-linking agent that is connected of steel.

Embodiment 4

With isopropyl alcohol and 9 sieves of 1 N nitric acid pretreatment, and at 70 ℃ with acutely jolted under (250rpm) in the PrSSPAABP solution 2% incubation 3 hours.Sieve was reacted 25 minutes with jolting down at 30 ℃ with TCEP (20mg/ml is in the 0.1M acetate buffer).To sieve and wash wash after, turn down reaction 1 hour with PEI-PDT (14IA-13-1) 30 ℃ and macroseism.The washing sieve also reacted 30 minutes with DTT (20mg/ml is in water) under similarity condition.At last, the sieve of washing was reacted 2 hours down with the 0.5ml Ad-GFP that can quick cracked cross-linking agent modifies the jolting of 30 ℃ and 250rpm.

In brief, dilute the new Ad-GFP batch of materials of 350 μ l (5e12/ml), and be added in the cross-linking agent 2 (13IA-55-3) of the quick hydrolysis of 75 μ l 12.2mg/ml among the PBS with 350 μ l carbonate buffer (pH=9.3).Modify and at room temperature carried out 30 minutes, at last reactant mixture is carried out gel filtration with Ultragel A6.Assembling section 5-8ml, and be used for combination.

The sieve of washing is placed monolayer A10 (n=6) and the HEK 293 (n=3) that converges.Culture is taken a picture, analyze with fluorimetry after 20 and 72 hours then.

The result of A10 and HEK (Fig. 5) clearly illustrates that GFP transduction sharply increased (A10 be ca.10 doubly) between 20 and 72 hours, then is not like this by the situation of the viral connected system of affine conjugant mediation.These results show owing to Ad release (bridging agent t at room temperature takes place in the bridging agent hydrolysis _1/2It is 26 days).

Embodiment 5

With 6 rustless steel Velocity ^TMSupport (Cordis Corp) is cleaned with isopropyl alcohol, THF, chloroform (following 2 hours at 55 ℃), 1N nitric acid (1 hour), and is heated to 260 ℃, keeps 1 hour.Then with support twisting on conduit, and with the reaction 4 hours under 58 ℃ and 250rpm of 2% PrSSPAABP aqueous solution.Then, with TCEP (25mg/ml is in the 0.1M acetate buffer) under 40-35 ℃ and 250rpm with sample reductase 12 5 minutes.After the washing, support is spent the night under 28 ℃ and 250rpm with PEI-PDT amplification agent (14IA-13-1) reaction.

Then, with acetate buffer and PBS washing sample, make it reaction 25 minutes under 28 ℃ and 250rpm with DTT 16mg/ml.Abreast, dilute 350 μ l Ad-GFP (5e12/ml), and be added in cross-linking agent 2 (13IA-55-3) solution of the quick hydrolysis of 75 μ l 12.2mg/ml among the PBS with 350 μ l carbonate buffer (pH=9.3).Modify and at room temperature carried out 30 minutes, at last reactant mixture is carried out gel filtration with Ultragel A6.Assembling section (5-8ml).28 ℃ with 250rpm under make the support of DTT step after scouring contact Ad/ cross-linking agent mixture 5 hours.

Use the method for having set up to make 6 rats accept stent graft with TEFLON protecting tube sheath.4 days execution animals behind the placing rack.With formalin perfusion fixing after, take out the artery segment of putting into support.Remove support, tremulous pulse is washed in PBS, and it is embedded in the complex (cold block prepares the mixture of normally used PVA and PEG among the histology) of optimum Cutting temperature.Cutting cube, the tremulous pulse section is directly carried out immunostaining through fluorescence microscopy or with anti-GFP antibody.In whole 6 rats, put into behind the support and all to observe strong GFP at the film of the artery segment of putting into support and adventitia in 3 days and express.

Embodiment 6

Design this experiment to study the rate of release of the Ad that directly is connected with the steel of poly-bisphosphonate modification.Use the hydrolysis dynamics of describing among the embodiment 2 and 3 and two kinds of different hydrolytic crosslinking agent (promptly being respectively cross-linking agent 1 (SHC) and cross-linking agent 2 (RHC)) of hydrolysis dynamics fast at a slow speed of having.In addition, the cross-linking agent RHC with two variable concentrations modifies Ad.Visible and make fluoremetry evaluation and microscopic evaluation become possibility for the Ad vision that the surface go up to be connected, with cross-linking agent and the co-modified Ad of fluorescence labels Cy3.

The release of the Ad of surface combination only with its with the hydrolysis key complete rupture that is connected of surface after just generation.Because the t of each single ester bond in the cross-linking agent _1/2All be the significantly higher of constant and RHC, so expect 1) crosslinker concentration increases because the time lengthening that the particulate whole cross-linker molecules hydrolysis of control single virus are needed, so will slow down the rate of release of Ad, with 2) to compare with Ad that SHC handles, the rate of release of the Ad that modifies with RHC will be faster.

For research discharges, use two kinds of methods: supernatant method described below and surperficial fluorescence associated method based on fluorescence.

The supernatant method

Under 37 ℃, will place leachate (PBS/0.06% tween 20) respectively in conjunction with the stainless steel foil of virus.Collect supernatant at preset time point, add new PBS/0.06% tween 20.Measure the amount of the Ad of Cy3 labelling in the supernatant with fluorimetry.

Surface fluorescence associated method

Virus in conjunction with after, use fluorescence microscopy paper tinsel surface immediately, use the standardization setting of microscope and camera to obtain four of every paper tinsel feeble field images at random.Then paper tinsel is placed the PBS/0.06% tween 20.Select at the fixed time and obtain image (with changing buffer and carrying out the supernatant sampling simultaneously) once more.The histogrammic average canbdle power analysing digital image that uses Adobe Photoshop to generate is used for the quantitative of the surperficial Ad that connects.

Use Carver press that the stainless steel foil of 14 1.25cm * 1.25cm is flattened, weigh, clean with isopropyl alcohol and nitric acid, single at 1%PrSSPAABP solution (N=12) or 1.5%PAABP solution (N=2, the contrast) in the reaction 3.5 hours (72 ℃, 200rpm).After a large amount of DDW washings, (20mg/ml is in the 0.1M acetate buffer for the sample contact TCEP that uses PrSSPAABP to handle; 37 ℃, 200rpm) 25 minutes.React then with the paper tinsel washing, and with PEI-PDT amplification agent (15IA-36-1) (90min, 37 ℃, 200rpm).Wash paper tinsel then with water, and react (15mg/ml with DTT; 30min, RT slightly jolts; 4 ℃ of following 30min).Quick washing paper tinsel, and with the co-modified Ad-tPA reaction of cross-linking agent of the Cy3/ cleavable of preparation as described below.

Virus (4.3 * 10 with trisection part 500 μ l ¹²/ ml) every part of usefulness 200 μ l carbonate-bicarbonate buffers (pH=9.3) are diluted to 700 μ l.Cross-linking agent 2 (the 13IA-55-3 that add 150 μ l Cy3NHS (1mg/ml is in the carbonate buffer) and 75 μ l cleavables then in the aliquot in trisection part virus (preparation #1); 20mg/ml is in the carbonate buffer).

Similarly prepare preparation #2, but replace the cross-linking agent of quick hydrolysis, using the cross-linking agent with long time hydrolysis dynamics of same molar ratio (the PBS solution of 75 μ l 21.4mg/ml) is cross-linking agent 2 (12IA-40-1).

At last, prepare preparation #3 similarly, but add the cross-linking agent 2 of 25 μ l rather than 75 μ l with preparation #1.For three kinds of Ad preparations, all under 30 ℃ and 200rpm in conjunction with 30 minutes, and in room temperature with do not jolt and time carried out again 20 minutes.Bonded Ad sample carries out purification with the pretreated Sepharose B6 of the PBS/3mM EDTA post of the degassing.Collect part, have 4-9ml (purification yield of preparation 1,2,3 is respectively ca.77,86 and 75%).

For obtaining the conversion factor between preparation fluorescence and the granule number, carry out with the relative Cy3 calibration trace of fluorimetry sample being measured immediately behind the spectrophotography.The average labelling degree of preparation 1,2,3 is respectively 2792,2472 and 4152 Cy3 residues of each virion.

Dilute preparation 1 and 2 with 1ml 0.36% tween/PBS, obtain tween/PBS based formulation of 6ml 0.06%.Dilute preparation 3 with tween/PBS of 1ml 0.36% and the tween of 1ml 0.06%/PBS, obtain tween/PBS based formulation of 7ml 0.06%.

With the aliquot preparation #1 (2.695 * 10 of 1ml ¹¹Granule) adds on the paper tinsel ##3-6.With the aliquot preparation #2 (3.01 * 10 of 1ml ¹¹Grain) adds on the paper tinsel ##7-10.With the aliquot preparation #3 (2.25 * 10 of 1ml ¹¹Grain) add that paper tinsel ##11-14 goes up and 2 (PAA PB-handles) contrast paper tinsel #1-2 on.Carried out under (200rpm) 13 hours with jolting at 28 ℃ in conjunction with (and pseudo-combination).With untapped residual virus preparation (2ml suspension 1 and 2,1ml suspension 3) contact with in conjunction with the identical condition of paper tinsel, contrast as on-consumable (depleted).Estimate the supernatant of on-consumable contrast and each paper tinsel then with fluorimetry and spectrophotography.At last, with each sample of fluorescence microscopy, every paper tinsel is got 4 presentation graphicses.Sample is placed bottle respectively, add 1ml 0.06% tween/PBS.At the preset time point, estimate paper tinsel supernatant and paper tinsel surface respectively by fluorimetry and fluorescence microscope.

Rate of release (the accumulation aliquot of the Ad that connects through the cross-linking agent 1 (SHC) of hydrolysis at a slow speed; Fig. 6) to put down manyly than the rate of release of the Ad that connects through the cross-linking agent 2 (RHC) of quick hydrolysis.In addition, the RHC of low concentration allows to discharge faster (curve is steeper) than the high concentration of this same chemical compound.Release (Fig. 7) by the surface fluorescence strength estimation parallel with these data on some degree (particularly early stage time point).But, the problem that has a current experiments design not take into full account.In the microscopy luminous decay that as if causes Cy3 on steel surface, this part damaged the release result's that this method obtains effectiveness.But owing to two experiments are all carried out under the same conditions, so this observation should not change total effect.

Embodiment 7

The amplification agent of in embodiment 1, describing, can also use following amplification agent in the amplification program based on polyallylamine.Being prepared as follows of this amplification agent described (seeing route 1).

By in aqueous solution, handling hydrochloric acid polyallylamine (PAAHCl, Sigma-Aldrich, number average molecular weight M with strongly basic anionite Dowex G-55 (Sigma, OH form) _n≈ 10kDa, weight average molecular weight Mw ≈ 15kDa, polymerization degree n ≈ 100) be converted into free PAA alkali.Then water is changed to the 2-propanol, uses the PAA aqueous slkali in the 2-propanol (to contain ca.1.1mmol/g NH ₂Group) is used for further synthesizing.With this solution (2.207g, 2.43mmol NH ₂) use CH ₂Cl ₂(5ml) dilution in cooled on ice, dropwise is added in CH under agitation 2 minutes ₂Cl ₂In SPDP (Pierce, 0.379g, 1.21mmol) solution.With mixture restir on ice 20 minutes, and disposable adding succinic anhydrides (Sigma-Aldrich, 0.161g, 1.61mmol).At restir on ice after 1 hour, with the mixture vacuum drying, with polymer residues and ethyl acetate coevaporation until curing.Solid polymer is washed with ethyl acetate, dry and be dissolved in and be added with KHCO ₃(0.556g is in water 555mmol) (10ml).Solution is filtered, use H ₃PO ₄Be acidified to pH=3.Leach polymer precipitation, water, ethyl acetate washing, vacuum drying.Yield 0.510g. ¹H NMR (D ₂O+K ₂CO ₃, pH=9) show with the modification of 2-pyridine disulfide group with succinyl ester group NHCOCH ₂CH ₂The modification of COOH (k ≈ 50) is almost equal, and 3 bands appear in the former signal, are positioned at δ 8.20,7.48 and 7.02ppm (intensity 1: 2: 1), the latter's CH ₂Be positioned at δ 2.41ppm.

Route 1

Though with reference to specific embodiment of the present invention the present invention is described in detail, those skilled in the art can make variations and modifications with clear to it under prerequisite without departing from the spirit and scope of the present invention.

Claims

1. compositions that is used for biomaterial is delivered to zooblast or tissue, described compositions comprises:

(a) biomaterial;

(b) has the biodegradable cross-linker part of hydrolysis key, wherein said biodegradable cross-linker part and described biomaterial covalent bond; With

(c) substrate, wherein said substrate and described biodegradable cross-linker part covalent bond, condition is that described biodegradable cross-linker is suitable for hydrolysis by the described hydrolysis key that ruptures, thereby discharges and send described biomaterial.

2. the compositions of claim 1, wherein said biomaterial is the member who is selected from nucleic acid, genophore, protein, peptide and cell.

3. the compositions of claim 1, wherein said biomaterial comprises medicine.

4. the compositions of claim 1, wherein said hydrolysis key comprises the acyl-oxygen key.

5. the compositions of claim 4, wherein said biodegradable cross-linker partly is to be selected from following member

6. the compositions of claim 1, wherein said substrate is the member who is selected from metal, metal-oxide, mineral, pottery, polymer, carbon, organise material and metallo organic material.

7. the compositions of claim 1, the wherein described biodegradable cross-linker some effects of Xuan Zeing time of being enough to discharge and send described biomaterial.

8. the compositions of claim 1, wherein said biomaterial comprises viral vector, described biodegradable cross-linker part is by representing with following formula (b):

And described substrate comprises metal.

9. the using method of the compositions of claim 1, described method comprises:

Make the compositions of claim 1 contact a period of time with described zooblast or described tissue, the described time is enough to make the hydrolysis of described hydrolysis key and discharges described biomaterial, thereby described biomaterial is delivered to described zooblast or described tissue.

10. the method for claim 9 wherein selects described biodegradable cross-linker part to influence the described time.

11. the preparation of compositions method of claim 1, described method comprises:

Biodegradable cross-linker is provided, and it has the biodegradable cross-linker part that (a) contains the hydrolysis key, (b) biomaterial reaction end group and (c) substrate reaction end group;

Substrate with at least one reactive group is provided;

Biomaterial is provided;

The substrate reaction end group reaction that makes described substrate and described biodegradable cross-linker is so that described biodegradable cross-linker part is covalently bound with described substrate; With

Make the biomaterial reactive end radical reaction of described biomaterial and described biodegradable cross-linker, thereby make described biomaterial and described biodegradable cross-linker part covalently bound to prepare described compositions.

12. the method for claim 11, the wherein said biomaterial reactive end radical reaction that makes described biomaterial and described biodegradable cross-linker is before the described substrate reaction end group reaction that makes described substrate and described biodegradable cross-linker or carry out simultaneously with it.

13. the method for claim 11, wherein said substrate reaction end group is the thiol reactant base.

14. the method for claim 11, wherein said biomaterial reaction end group are at least a in sulfosuccinimide base ester group, trifluoro esilate group and the epoxide group.

15. the method for claim 11, at least one reactive group of wherein said substrate is a mercapto.

16. the method for claim 11, wherein said biodegradable cross-linker are to be selected from following member

17. the method for claim 11, wherein said biomaterial are the members who is selected from nucleic acid, genophore, protein, peptide and cell.

18. the method for claim 11, wherein said biomaterial comprises medicine.

19. the method for claim 11, wherein said substrate are the members who is selected from metal, metal-oxide, mineral, pottery, polymer, carbon, organise material and metallo organic material.

20. the method for claim 11, wherein said biomaterial and the reaction before making the reaction of described substrate and described substrate reaction end group of described biomaterial reaction end group form the biomaterial that biodegradable cross-linker is modified.

21. the method for claim 20 wherein saidly provides the reaction of described biodegradable cross-linker and described and described biomaterial to comprise:

(i) is provided first reactant, it has the described biomaterial reaction end group and first end functional groups, (ii) second reactant, its comprise described biodegradable cross-linker part that (a) contain described hydrolysis key, (b) can with second end functional groups of described first end functional groups reaction and (c) substrate reaction end group;

Make the biomaterial reactive end radical reaction of described biomaterial and described first reactant; With

Make first functional group and second functional group reaction, form the biomaterial that described biodegradable cross-linker is modified.

22. the method for claim 21, wherein said first reactant is maleimide-(sulfo group) succinimido ester, maleimide-trifluoro esilate or pyridine disulfide group-(sulfo group) succinimido ester, and described second reactant is dithiol, mercaptan-dimethyl sulfide or two (dimethyl sulfide).

23. one kind is delivered to the method for zooblast or tissue with biomaterial, described method comprises:

A kind of compositions is provided, and it comprises (a) biomaterial; (b) has the biodegradable cross-linker part of hydrolysis key, wherein said biodegradable cross-linker part and described biomaterial covalent bond; (c) substrate, wherein said substrate and described biodegradable cross-linker part covalent bond, condition is that described biodegradable cross-linker is suitable for hydrolysis by the described hydrolysis key that ruptures, thereby discharges described biomaterial; With

Make described compositions contact a period of time with zooblast or tissue, this time is enough to make the hydrolysis of described hydrolysis key and discharges described biomaterial, thereby described biomaterial is delivered to described zooblast or described tissue.